|
| Status |
Public on Apr 04, 2024 |
| Title |
RNAseq_E135_exColon_EPCAM_mCherry_Trp53KO_Rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Colon endoderm
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Colon endoderm
developmental stage: E13.5
strain: mCherry+ morula (CD1/G4) aggregated with GFP+ mESCs (G4)
genotype: Trp53 KO
|
| Treatment protocol |
Embryos were generated by diploid complementation assay with aggregation. In each depression of the aggregation plate, one small GFP+ mESC colony of about 8-15 cells was added together with one zona pellucida-free mCherry+ pre-compaction morula.
|
| Growth protocol |
GFP+ mESCs were cultured on mitotically inactive primary mouse embryo fibroblasts in serum/LIF condition for two days. Aggregated embryos, cultured in KSOM, that progressed to the expanded blastocyst stage were transferred to a pseudo-pregnant CD1 female. Post-implantation embryos were isolated at the indicated stage, including a 24-hour developmental delay due to the uterine transfer procedure.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Extraembryonic tissues were removed, the intestine was dissected from a single embryo, and split into the small intestine and colon parts with a micro knife. The colon was dissociated using 0,25% Trypsin-EDTA, at 37 ºC for 10 minutes. The cells were washed with 10% FBS/DMEM, stained first with EPCAM antibody then with DAPI in 2% FBS/HBSS with 0,5 mM EDTA (FACS buffer) at 4 ºC for 10 and 8 minutes, respectively. Cells were resuspended in FACS buffer, the indicated populations were sorted into Buffer RLT plus using the BD FACSAria Fusion, and the samples were stored at -80 ºC.
RNA-seq libraries were prepared as part of the Smart-RRBS protocol by Gu, H et al. 2021 Nature Protocols, with the adaptation to low input bulk samples instead of single-cell input by reducing the cDNA amplification cycle number according to the input.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Raw reads were subjected to adapter and quality trimming with cutadapt (version 4.1; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25 --interleaved --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC), followed by poly-A trimming with cutadapt (parameters: --interleaved --overlap 20 --minimum-length --adapter "A[100]" --adapter "T[100]").
Reads were aligned to the mouse reference genome (mm10) using STAR (version 2.7.9a; parameters: --runMode alignReads --chimSegmentMin 20 --outSAMstrandField intronMotif --quantMode GeneCounts) and transcripts were quantified using stringtie (version 2.0.6; parameters: -e) with GENCODE annotation (release VM23).
Assembly: mm10 (including mCherry and GFP transgenes)
Supplementary files format and content: Tab-delimited file containing gene abundances: <Gene ID> <Gene Name> <Reference> <Strand> <Start> <End> <Coverage> <FPKM> <TPM>
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| |
|
| Submission date |
Dec 13, 2023 |
| Last update date |
Apr 04, 2024 |
| Contact name |
Sara Hetzel |
| E-mail(s) |
hetzel@molgen.mpg.de
|
| Organization name |
Max Planck Institute for Molecular Genetics
|
| Department |
Genome Regulation
|
| Lab |
Meissner Lab
|
| Street address |
Ihnestraße 63
|
| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE250083 |
Extraembryonic gut endoderm cells undergo programmed cell death during development (RNA-seq) |
| GSE250084 |
Extraembryonic gut endoderm cells undergo programmed cell death during development |
|
| Relations |
| BioSample |
SAMN38811573 |
| SRA |
SRX22879120 |
